The goal of this proposal is to investigate how the essential metabolite acetyl-coenzyme A (Ac-CoA) links cellular metabolism to changes in gene expression. The lysine acetyltransferases (KATs) general control nonderepressible 5 (GCN5) and p300/CBP-associated factor (PCAF) are global transcription factors that function in multisubunit complexes to acetylate histone and non-histone proteins to regulate gene transcription. As a necessary cofactor for KAT activity, Ac-CoA cellular concentration is directly related to increased rates of GCN5 and PCAF activity, protein target acetylation, and the transcriptional activation of growth-promoting genes. In metazoans, Ac-CoA is produced from glucose-derived citrate by the enzyme adenosine triphosphate (ATP)-citrate lyase (ACLY). Our collaborator, Dr. Kathryn Wellen, has reported that ACLY links cellular metabolism to histone acetylation, with ACLY-dependent production of Ac-CoA driving increased histone acetylation. In addition, many actively-proliferating cells exhibit increased levels of ACLY, and the proliferation of cancer cells in particular is sensitive to the availability of glucose and proper ACLY function. ACLY also undergoes an increase in stability in response to elevated glucose levels and activation of growth-promoting signaling pathways by KAT-dependent transcription activation. Moreover, new unpublished data from the Wellen laboratory suggest that ACLY and PCAF form a complex in cells, complementing a recent report demonstrating that PCAF acetylates ACLY to promote ACLY stability and tumor growth. Yet, the nature of the communication between ACLY and GCN5/PCAF and how this communication may regulate the activity of each of the enzymes is not known. Based on these data, this proposal seeks to test the hypothesis that ACLY forms a direct complex with the GCN5 and PCAF to facilitate both ACLY acetylation and stability and GCN5 and PCAF acetylation activity towards histone substrates, to support the activation of growth promoting genes. This hypothesis will be investigated using in vitro pull-down assays on biochemically purified ACLY and GCN5/PCAF, in order to determine if they can bind directly to one another. The biophysical properties of the interaction will then be characterized with ITC, as well as the steady state kinetic parameters of the interacting enzymes by enzyme activity assays. Lastly, X-ray crystallography structure determination and mutagenesis experiments will provide the molecular basis for the interaction of ACLY with GCN5/PCAF. Lastly, this proposal will determine the in vivo consequence of the ACLY-GCN5/PCAF interaction by disrupting this interaction and interrogating mammalian cell lines for changes in global histone acetylation, the expression of specific metabolism and growth-promoting genes, and the ability of these cells to proliferate and differentiate. Together, these studies will delineate the molecular mechanism for how the AcCoA metabolite links cellular metabolism to gene expression and will provide new avenues for targeting ACLY function for cancer therapy.